Tissue specific variation of C-glycosylflavone patterns in oat leaves as influenced by the environment

A comparison is made between the flavone patterns accumulating in epidermal tissues and in the mesophyll of oat primary leaves grown in a phytotron and under field conditions. In developing leaves cultivated under standard conditions, varying patterns of two vitexin-derived O-rhamnosides and of one isovitexin O-arabinoside are produced in the basal region as the result of basal meristem activity. These patterns are tissue specific and differ quantitatively in the epidermis and the mesophyll. During the course of subsequent growth and differentiation, this pattern is constant as the compounds are moved upwards due to basipetal leaf growth. In comparison, the flavone patterns generated in the basal section of leaves grown in the field do not vary significantly. There is the additional accumulation of isoorientin O-arabinoside. Again flavone patterns are tissue specific, but in contrast to standard growth they are modified characteristically in those leaf tissues which are already morphologically differentiated. It is possible that the isovitexin moiety of the O-arabinoside is oxidized to the corresponding isoorientin derivative in the mesophyll. Moreover, field-grown leaves show a two-fold increase in flavone content in each leaf epidermis and a six-fold increase in the mesophyll when compared to the corresponding tissues of phytotron-grown leaves.     

Soil-borne microorganisms and soil-type affect pyrrolizidine alkaloids in Jacobaea vulgaris

Secondary metabolites like pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. We studied the effects of soil-borne microorganisms and soil-type on pyrrolizidine alkaloids in roots and shoots of Jacobaea vulgaris. We used clones of two genotypes from a dune area (Meijendel), propagated by tissue culture and grown on two sterilized soils and sterilized soils inoculated with 5% of non-sterilized soil of either of the two soil-types. Soil-borne microorganisms and soil-type affected the composition of PAs. By changing the composition rather than the total concentration below and aboveground, plants have a more complex defense strategy than formerly thought. Interestingly, a stronger negative effect on plant growth was found in sterilized soils inoculated with their ‘own’ microbial community suggesting that pathogenic and/or other plant inhibiting microorganisms were adapted to their ‘own’ soil conditions.     

Light as a regulator of structural and chemical leaf defenses against insects in two Prunus species

Light is a key factor influencing competition between species, and the mechanisms by which trees overcome insect outbreaks can be associated with alternation of the leaves structure, which then prevent or promotes their susceptibility to herbivores. It was predicted that leaf tissue anatomy would likely be different in sun and shade leaves, with a gradual decline of leaves resistance coupled with reduction of accessible light. We quantified anatomical patterns and the distribution of defence compounds (phenols, total tannins, catechol tannins) within heavily grazed leaves of Prunus padus, native in Europe and Prunus serotina, an invasive to Central Europe. Both species were strongly attacked by folivorous insects when shrubs grew in the shade. In the sun, however only P. padus leaves were grazed, but P. serotina leaves were almost unaffected. We identified that anatomical characteristics are not linked to different P. padus and P. serotina leaf vulnerability to insects. Furthermore, the staining of defence compounds of P. serotina leaves grown in full sun revealed that the palisade mesophyll cells had a higher content of phenolic compounds and catechol tannins. Thus, our results indicate that a specific distribution of defence compounds, but not the anatomical relationships between palisade and spongy mesophyll, may be beneficial for P. serotina growth outside its natural range. The identified pattern of defence compounds distribution is linked to a lower susceptibility of P. serotina leaves to herbivores, and is associated with its invasiveness. This likely reflects that P. serotina is a stronger competitor than P. padus, especially at high sunlit sites i.e. gaps in the forest.     

Attract and deter: a dual role for pyrrolizidine alkaloids in plant–insect interactions

Pyrrolizidine alkaloids (PAs) are the major defense compounds of plants in the Senecio genus. Here I will review the effects of PAs in Senecio on the preference and performance of specialist and generalist insect herbivores. Specialist herbivores have evolved adaptation to PAs in their host plant. They can use the alkaloids as cue to find their host plant and often they sequester PAs for their own defense against predators. Generalists, on the other hand, can be deterred by PAs. PAs can also affect survival of generalist herbivores. Usually generalist insects avoid feeding on young Senecio leaves, which contain a high concentration of alkaloids. Structurally related PAs can differ in their effects on insect herbivores, some are more toxic than others. The differences in effects of PAs on specialist and generalists could lead to opposing selection on PAs, which may maintain the genetic diversity in PA concentration and composition in Senecio species.     

Wetland ecosystem comparison using a suite of plant assessment measures

In light of extensive human impact on wetlands it is necessary that we develop an effective way to monitor the effects of impact in order to prevent further destruction. One method is plant community assessment, specifically Floristic Quality Assessment (FQA), which is common, but can be subjective. In this case study, we implement FQA, as well as specific morphological and chemical assessment measures over a two-year period in order to compare two wetlands in the Lake George watershed in the Adirondack mountains and their response to human impact. While the wetlands studied demonstrated very different water chemistry profiles makeups, FQA did not reveal substantial differences between plant communities. However, more specific analyses of plant morphology and tissue chemistry did reveal significant differences that reflected the level of impact at these two sites. Namely, the simple plant Lemna minor had consistently shorter roots and Nuphar lutea contained higher amounts of nitrogen in above ground tissues when growing in an anthropogenically impacted wetland. We suggest that FQA and specific plant morphology and tissue chemistry measurements be performed concurrently to provide indication of both long- and short-term effects of human impact in wetland ecosystems.     

Metabolic engineering and potential functions of proanthocyanidins in poplar

Poplar (Populus spp.) is a widely distributed tree genus of significant economic and ecological importance. Poplar trees accumulate proanthocyanidins (PAs) in leaves, roots, and a variety of other tissues. Damage to leaves by insects causes a rapid accumulation of PAs, both at the site of damage and distally in undamaged leaves. This rapid PA accumulation is mediated by the activation of genes encoding enzymes involved in PA synthesis. PAs have been hypothesized to deter insect feeding and reduce the nutritive value of poplar leaf tissue, but experimental evidence supporting a role for PAs as an effective inducible defense against herbivores is lacking. Our recent paper described the identification of a MYB gene that regulates the PA pathway under multiple stress conditions, and we used this gene to constitutively activate the PA pathway in poplar. Here we describe observations that suggest that poplar PAs may have roles besides insect defense, for example, responses to UV light. The PA-modified trees will be a useful tool for analyzing the biological roles of PAs in this important model tree.Key words: tannins, herbivory, flavonoid, UV light, light stress     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Frequent gain and loss of pyrrolizidine alkaloids in the evolution of Senecio section Jacobaea (Asteraceae)

Pyrrolizidine alkaloids (PAs) of the macrocyclic senecionine type are secondary metabolites characteristic for most species of the genus Senecio (Asteraceae). These PAs are deterrent and toxic to most vertebrates and insects and provide plants with a chemical defense against herbivores. We studied the PA composition of 24 out of 26 species of Senecio section Jacobaea using GC-MS. The PA profiles of eight of these species have not been studied before and additional PAs were identified for most other species that were included in previous studies. With one exception (senecivernine) all 26 PAs identified in sect. Jacobaea can be regarded as derivatives of the biosynthetic backbone structure senecionine. Based on the PA profiles of the species of sect. Jacobaea and the results of previous tracer studies, we constructed two hypothetical biosynthetic scenarios of senecionine diversification. Both scenarios contain two major reactions: the conversion of the necine base moiety retronecine into the otonecine moiety and site-specific epoxidations within the necic acid moiety. Further reactions are site-specific hydroxylations, sometimes followed by O-acetylations, site-specific dehydrogenations, E, Z-isomerizations, and epoxide hydrolysis and chlorolysis. The GC-MS data and both biosynthetic scenarios were subsequently used to study the evolution of PA formation in sect. Jacobaea by reconstructing the evolutionary history of qualitative PA variation in this section. This was achieved by optimizing additive presence/absence data of PAs and types of enzymatic conversions on a maximum parsimony cladogram of section Jacobaea inferred from DNA sequence and morphological data. Besides showing large intra- and interspecific variation, PA distribution appears to be largely incidental within the whole clade. These results together with the finding that all but one of the PAs identified in sect. Jacobaea are also present in species of other sections of Senecio indicate that differences in PA profiles in Senecio can not be explained by the gain and loss of PA specific genes, but rather by a transient switch-off and switch-on of the expression of genes encoding PA pathway-specific enzymes.     

The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine - chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F(2) hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA%=the tertiary amine concentration/(tertiary amine concentration+the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.     

The correlation between leaf-surface and leaf-tissue secondary metabolites: a case study with pyrrolizidine alkaloids in <Emphasis Type="Italic">Jacobaea</Emphasis> hybrid plants

Introduction

Usually whole plant or whole leaf extracts are analyzed to study the chemical ecology of insect-plant interactions. For herbivore species the contact with the leaf surface enables them to estimate the quality of the plant. The relationship between the leaf-surface and leaf-tissue secondary metabolites (SMs) could offer important new insights in insect-plant interactions mediated by SMs. Pyrrolizidine alkaloids (PAs), typical defense chemicals in Jacobaea species, are repellent for generalist herbivores but are attractive to specialists.

Objectives

Explore whether the PAs on the leaf surface are a reliable representation of the PAs in the leaf tissue in PA-containing plants.

Method

The concentration of individual PAs present on the leaf surface and in the corresponding leaf tissue from 37 genotypes (one plant from each genotype) of an F₂ generation of a cross between Jacobaea vulgaris and Jacobaea aquatica was measured by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). PAs were removed from the leaf surface by extraction with a slightly acidic aqueous solution.

Results

The total amount of PAs present on the surface of the leaves was only 0.015% (range 0.001–0.163%) of the total amount present in the leaf tissue. Most PAs present in the leaf tissue were also found on the surface, except for jaconine, dehydrojaconine, dehydrojacoline and usaramine N-oxide. Positive correlations between leaf-surface and leaf-tissue concentrations were found for most of the jacobine-like and otosenine-like PAs, but correlations for total PA, senecionine- and erucifoline-like PAs were not significant.

Conclusion

These results indicate that PA variation on the leaf surface only partially reflects the PA variation in the leaf tissue. Because most herbivores are affected in a different manner by individual PAs, this result means that the leaf surface does not give a reliable estimate of plant quality to herbivores.

     

Effects of pyrrolizidine alkaloids through different trophic levels

Pyrrolizidine alkaloids (PAs), mainly those with a 1,2-double bond in the necine base moiety (=1,2-dehydropyrrolizidines), constitute a class of well studied compounds with respect to their flux through different trophic levels. Plants belonging to various clades (e.g. Echiteae, Eupatorieae and Senecioneae, Boraginaceae, and Crotalarieae) biosynthesize PAs as N-oxides, generally in the roots, and transport them through the phloem to stems, leaves, and reproductive structures, where they act as potent deterrents against non-specialist herbivores. On the other hand, PA specialist herbivores (mainly arctiid moths, danaine and ithomiine butterflies, and some leaf beetles) have become able to overcome this chemical barrier, and to sequester these alkaloids from their larval host plants or from sources visited by adults, such as flowers and dead or withered plants. Specialists use PAs for their own benefit as chemical defence against a vast array of predators (e.g. ants, lacewings, spiders, lizards, birds, and mammals), but some predators are able to feed on PA-insects, by avoiding or physiologically overcoming PAs present in tissues of the ingested prey. Parasitoids may be affected by PAs, depending on their degree of specialization in relation to PA-insects. Arctiidae, Danainae and Ithomiinae also use PAs as precursors of sexual pheromones. The effects of PAs on trophic interactions have been intensely studied over the last four decades, but some open questions remain, and are discussed, such as the underlying mechanisms that lead to PA diversification, activity of different PA structures, synergism among PAs and other so-called defensive substances in PA-plants, and the ability to overcome this chemical barrier by predators and parasitoids.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant’s constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitments occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a “processed pseudogene”, and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.     

Biochemical characterization of cell types within leaves of metal-hyperaccumulating Noccaea praecox (Brassicaceae)

Background and aims

Distinct metal distribution patterns within leaves of metal hyperaccumulating plants are repeatedly observed however, the presumable role of key structural biochemical molecules in determining and regulating their allocation remains largely unknown. We aimed to characterise in a spatially resolved manner the distribution of the main biochemical components in leaves of field-collected Cd/Zn-hyperaccumulating Noccaea praecox in order to relate them to metal distribution patterns at tissue level.

Methods

The biomolecular composition of the leaves was spatially analysed using synchrotron radiation Fourier Transform Infrared (FTIR) and the distribution of Zn with synchrotron radiation Low-Energy X-Ray Fluorescence (LEXRF) microspectroscopy was determined on the same tissues of interest (epidermis, sub-epidermis, mesophyll).

Results

In epidermal cells high proportion of free-carboxyl, nitro and phosphate groups standing for pectin, nitroaromatics, phytic and other organic acids were found. Adjacent mesophyll cells had higher proportions of proteins, carbohydrates and cellulosic compounds.

Conclusions

Pectin compounds were indicated as important components of Zn enriched epidermal cell walls. In addition, intense lignification of epidermal cell walls might limit leakage of the trapped metals back to the metabolically active and thus more sensitive mesophyll. Distribution of metal-binding compounds in particular cell types/tissues may therefore predispose metal distribution patterns and tolerance in leaves.     

Systematic analysis of rice (Oryza sativa) metabolic responses to herbivory

Plants defend against attack from herbivores by direct and indirect defence mechanisms mediated by the accumulation of phytoalexins and release of volatile signals, respectively. While the defensive arsenals of some plants, such as tobacco and Arabidopsis are well known, most of rice's (Oryza sativa) defence metabolites and their effectiveness against herbivores remain uncharacterized. Here, we used a non‐biassed metabolomics approach to identify many novel herbivory‐regulated metabolic signatures in rice. Most were up‐regulated by herbivore attack while only a few were suppressed. Two of the most prominent up‐regulated signatures were characterized as phenolamides (PAs), p‐coumaroylputrescine and feruloylputrescine. PAs accumulated in response to attack by both chewing insects, i.e. feeding of the lawn armyworm (Spodoptera mauritia) and the rice skipper (Parnara guttata) larvae, and the attack of the sucking insect, the brown planthopper (Nilaparvata lugens, BPH). In bioassays, BPH insects feeding on 15% sugar solution containing p‐coumaroylputrescine or feruloylputrescine, at concentrations similar to those elicited by heavy BPH attack in rice, had a higher mortality compared to those feeding on sugar diet alone. Our results highlight PAs as a rapidly expanding new group of plant defence metabolites that are elicited by herbivore attack, and deter herbivores in rice and other plants.     

On the Resistance to Transpiration of the Sites of Evaporation within the Leaf

The rates of transpiration from the upper and lower surfaces of leaves of Gossypium hirsutum, Xanthium strumarium, and Zea mays were compared with the rates at which helium diffused across those leaves. There was no evidence for effects of CO₂ concentration or rate of evaporation on the resistance to water loss from the evaporating surface (“resistance of the mesophyll wall to transpiration”) and no evidence for any significant wall resistance in turgid tissues. The possible existence of a wall resistance was also tested in leaves of Commelina communis and Tulipa gesneriana whose epidermis could be easily peeled. Only when an epidermis was removed from a leaf, evaporation from the mesophyll tissue declined. We conclude that under conditions relevant to studies of stomatal behavior, the water vapor pressure at the sites of evaporation is equal to the saturation vapor pressure.